Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

Induction of suppressor cells to T- and B-cell proliferative responses and immunoglobulin production by monoclonal antibodies recognizing the CD3 T-cell differentiation antigen

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.     

Regulatory interactions governing the proliferation of T cell subsets stimulated with pokeweed mitogen

Serial phenotyping of human peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM) demonstrated an excess of T8+ cells after stimulation. Preferential expansion of the T8+ cell compartment was a result of T8+ cell blast transformation while T4+ cells generated fewer blasts and tended to remain as small resting cells. When the proliferative behavior of T cell subsets in PWM-stimulated PBMC with physiologic proportions of T4+ and T8+ cells was compared with that of cultures depleted of T4+ or T8+ cells, two levels of regulation of proliferation were found: without T4+ cell help, T8+ cells were unable to divide; however, in the presence of T4+ cells, PWM-stimulated T8+ cells became potent feedback inhibitors of T4+ cell proliferation. The mechanism of suppression by PWM-activated T8+ cells of T4+ cell proliferation, not only to PWM, but also to tetanus toxoid, was pursued by measuring decreased interleukin 2 (IL2) recovery from cultures containing suppressors. Although passive absorption of IL2 by PWM-activated cells could contribute to the suppression of fresh proliferative responses, as shown directly with isolated T4+ cells induced by PWM to express IL2 receptors, a much more profound suppression was mediated by PWM-activated T8+ cells. The regulation of proliferative responses of helper and suppressor T cell subsets may determine the magnitude of their subsequent interactions and thus control the ultimate outcome of in vivo physiologic and pathologic immune responses.     

Immunofluorescence detection of delta opioid receptors (DOR) on human peripheral blood CD4+ T cells and DOR-dependent suppression of HIV-1 expression.

The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-([2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,-,(2S[1(S*),2alpha,5beta])-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 +/- 0.70 (mean +/- SEM) to 20.70 +/- 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed (approximately 50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.     

Functional heterogeneity among CD4+ encephalitogenic T cells in recruitment of CD8+ T cells in experimental autoimmune encephalomyelitis

Inoculation of Lewis rats with live or attenuated (irradiated or paraformaldehyde-fixed) CD4+ encephalitogenic T cells (S1 line) protects the recipients from transferred experimental autoimmune encephalomyelitis (tEAE) induced by S1 cells. A CD8+ T lymphocyte population specifically activated against the EAE-inducing S1 cells can be readily isolated from the lymphoid organs of pretreated animals. We show, in the present study, that encephalitogenic T cell lines derived from Lewis rats differ in their ability to induce resistance against tEAE in vivo and to stimulate CD8+ cell proliferation in vitro. We also demonstrate that the S19 line of encephalitogenic T cells, in combination with myelin basic protein (MBP), can stimulate CD8+ cell proliferation in vitro. The CD8+ cells generated in this way strongly suppress MBP-specific T cell proliferation in vitro. This combined effect of T cells and MBP was also evident in vivo. Neither S19 cells nor MBP alone induced resistance against S19-mediated tEAE, rather coinjection of these cells and MBP was required. Our results suggest that resistance to EAE is mediated by distinct populations of encephalitogenic T cells that activate Ts cells through different mechanisms. In some instances, both autoreactive T cells and their relevant autoantigen(s) may be needed to activate Ts cells in vivo.     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Promotion of human T lymphocyte proliferation by IL-4

The capacity of human rIL-4 to support the proliferation of mitogen-stimulated T cells directly as well as by increasing IL-2 production or enhancing IL-2 responsiveness was investigated. IL-4 augmented proliferation of T cells stimulated with PHA, Con A, immobilized mAb to the CD3 molecular complex (OKT3), or PMA. IL-4 increased the number of mitogen-stimulated cells entering the cell cycle as well as enhancing ongoing proliferation of mitogen-activated lymphoblasts. Facilitation of initial activation by IL-4 was not inhibited by mAb to the p55 component of the IL-2R, anti-Tac, and, therefore, was not dependent on endogenous IL-2 activity. However, IL-4-mediated enhancement of ongoing T cell proliferation stimulated by PHA or OKT3 was partially but not completely blocked by anti-Tac. Analysis of the supernatants from PHA-stimulated T cell cultures indicated that IL-4 increased the production of IL-2 by mitogen-activated cells. Moreover, IL-4 increased the amount of IL-2 mRNA that accumulated in mitogen-stimulated T cells. In addition, IL-4 markedly augmented IL-2R expression by PHA-stimulated T cells. Although IL-4 promoted ongoing DNA synthesis of mitogen-stimulated T cells in an IL-2-dependent manner, it was also able to sustain their proliferation directly. Thus, IL-4 supported proliferation of PMA-activated T cells in a manner that was not inhibited by anti-Tac. Furthermore, IL-4 could augment proliferation and IL-2R expression of T cells stimulated with PHA in the presence of cyclosporin A, which blocks endogenous cytokine production or anti-Tac. Finally, IL-4 was noted to enhance proliferation of both CD4+ and CD8+ T cell subsets. The results indicate that IL-4 enhances proliferation of mitogen-activated human T cells by a number of mechanisms, including the direct promotion of cell cycle entry and subsequent DNA synthesis, enhanced production of IL-2, and increased responsiveness to IL-2 in part by up-regulation of IL-2R expression.     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

Immune regulation by platelet-activating factor. I. Induction of suppressor cell activity in human monocytes and CD8+ T cells and of helper cell activity in CD4+ T cells

Platelet-activating factor (PAF) is a powerful mediator of inflammation. We have recently described a potential role for PAF in immune reactions, as it inhibits T cell proliferation and IL-2 production in response to mitogens. To further define the mechanism through which this inhibition is exerted, we used a coculture system in which PBML are preincubated with increasing concentrations of PAF for 24 h, followed by washing, treatment with mitomycin C and addition to fresh autologous PBML stimulated with PHA. In this context, a significant (40 to 60%) inhibition of proliferation was observed. In parallel, PAF-pre-treated cells induced a reduction (30 to 50%) of IL-2 production by PHA-stimulated lymphocytes. The PAF receptor antagonist BN52021 could partially block the PAF-induced suppressor cell activity, but also showed some suppressor cell-inducing properties of its own (20 to 30%). The expression of suppressor cell function during the co-culture could be partially abrogated by the inclusion of indomethacin, suggesting that cycloxygenase metabolites of arachidonic acid were involved in this phase of suppression. When PBML were fractionated into monocytes, lymphocytes, or T cell subsets before pre-incubation with PAF, indomethacin-sensitive suppressor cell function was generated in the monocyte population. Monocyte-depleted lymphocytes showed slight helper effect, whereas CD8+ T cells were induced to become indomethacin-resistant suppressor cells. CD4+ T cells, in contrast, were activated to exert very marked helper effect. When incubated with PAF for 24 h, monocyte-depleted lymphocytes showed a 30% decrease in CD4+ T cell numbers and a 50% increase in CD8+ T cell numbers. Our data suggest a novel immunoregulatory role for PAF and potentially important interactions of this lipid mediator of inflammation with lymphocyte and monocyte functions.     

In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells

In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages. Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset. We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates. Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin. After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA. Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03). Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture. The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive. Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations. Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells. Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis. The data suggest that UV-exposure modulates cutaneous APC activity in humans, as in mice, such that the dominant immune response is tilted toward suppression. These mechanisms in normal individuals may function to dampen responses to UV-induced endogenous Ag that are pathogenic in autoimmune disorders. However, these mechanisms might also facilitate the growth of UV-induced skin cancers.     

Immunotherapy for visceral leishmaniasis: ability of factors produced during anti-leishmania responses of skin test positive adults to inhibit peripheral blood mononuclear cell activities associated with visceral leishmaniasis

The course of human Leishmania chagasi infections appears to be determined by the balance between type 1 (Tl) CD4+ and CD8+ T suppressor (Ts) cell activities. Skin test positive adults living in hyperendemic areas who have no history of visceral leishmaniasis (VL) have Tl CD4+ T cell immunodominant responses against L. chagasi. The cytokines they secrete during anti-leishmania responses are a probable source of cytokines which inhibit the CD8+ Ts cells associated with VL. The ability of supernatants generated from peripheral blood mononuclear cells derived from skin test positive adults to reverse immune responses which appear to be mediated by CD8+ Ts cells was assessed in three sets of screening assays. The supernatants displayed three candidate factors. One, which could be explained by Leishmania antigens in the supernatant, decreased high endogenous IL-10 secretion characteristic of one class of VL patients. A second activity decreased high endogenous proliferation characteristic of the same class of patients without decreasing antigen specific proliferation. The third activity inhibited or killed CD8+ T cells but not CD4+ T cells. These activities might be useful in treating VL.     

Development of self-tolerance in normal mice. Appearance of suppressor cells that maintain adult self-tolerance follows the neonatal autoantibody response

T cell populations from BALB/c mice at different ages were analyzed to determine when in development Ts cells specific for the anti-mouse RBC (MRBC) autoantibody response become activated. Previous studies have shown that adult CD8+ T cells actively suppress this autoimmune response and adult spleen cells depleted of CD8+ cells can generate an anti-MRBC response in culture with MRBC. The present results demonstrate that T cells from mice less than 1 wk of age do not suppress the in vitro anti-MRBC response of adult spleen cell populations depleted of CD8+ Ts cells. By 2 wk of age Ts cells are detectable in this anti-self response and reach adult levels by 3 wk of age. Non-specific "natural suppressor" cells normally present in neonatal spleen cell populations are unable to suppress this autoantibody response, although they are active in suppressing anti-SRBC responses in the same cultures. Before the appearance of Ts cells active in the anti-MRBC response, neonatal spleen cell populations can generate anti-MRBC antibody-forming cells, both spontaneously in vivo and in vitro. The in vitro anti-MRBC response of neonatal spleen cells was shown to be Ag driven and Ag specific. The ability of unfractionated spleen cells to generate this response in vitro declines with age and is relatively low by 3 wk. This decline in responsiveness occurs simultaneously with the appearance of suppression specific for the anti-MRBC response, suggesting that the two events may be causally related.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

Activation through CD3 molecule leads a number of human T cell clones to induce IgE synthesis in vitro by B cells from allergic and nonallergic individuals

Seventy-eight clones established from tonsillar T lymphocytes of two nonallergic children were tested under different experimental conditions for their ability to induce in vitro IgE synthesis by B cells from allergic or nonallergic donors. After 24 hr preactivation with phytohemagglutinin (PHA), 11 out of 32 CD4+ clones from the first and 17 out of 36 CD4+ clones from the second tonsil donor showed the ability to induce IgE synthesis in vitro by B cells from both allergic and nonallergic individuals, whereas none of 10 CD8+ clones nor T blasts of PHA-induced cell lines obtained from unfractionated T cell suspensions of the same tonsils had such an effect. Seven of the 11 T cell clones from the first tonsil donor active on IgE production after pre-activation with PHA also induced IgE synthesis in vitro by nonallergic and allergic B cells upon stimulation with anti-CD3 monoclonal antibody. Under the same experimental conditions, virtually all of the T cell clones able to induce IgE synthesis in vitro by target B cells showed the ability to stimulate IgG and IgM production as well. T cell clones were also established from the peripheral blood of a nonallergic donor and were tested for their ability to induce IgE synthesis in autologous B cells. After preactivation with PHA, seven out of 35 CD4+ clones induced the production of detectable amounts of both IgE and IgG in autologous B cells. The addition to the cultures of PHA-stimulated unfractionated T cells inhibited in a dose-dependent manner the IgE but not the IgG synthesis induced by an autologous helper T cell clone in autologous B cells. Taken together, these data indicate that a remarkable proportion of human T cell clones upon triggering of the CD3 molecular complex were able to provide help for the synthesis of IgE in B cells from both allergic and nonallergic individuals. The successful induction of IgE synthesis by single T cell clones was apparently related to the lack of concomitant suppressor activity to which IgE-producing cells appeared to be exquisitely sensitive.     

Differences in surface phenotype between cytolytic and non-cytolytic CD4+ T cells. MHC class II-specific cytotoxic T lymphocytes lack Leu 8 antigen and express CD2 in high density

A number of cell surface molecules are differentially expressed on functionally distinct subsets of CD4+ T cells. However, to date CD4+ T cells capable of becoming CTL have not been shown to be phenotypically distinct from other CD4+ T cells, and in the current study we examined the ability of Leu 8+ and Leu 8- CD4+ subpopulations to become cytotoxic effectors after their stimulation with allogeneic lymphoblastoid cell lines. Although CD4+, Leu 8+ cells proliferated more vigorously than CD4+, Leu 8- cells in primary cultures stimulated with allogeneic LCL, the CD4+, Leu 8- population was the major source of cytotoxic effectors, killing targets with specificity for their class II MHC alloantigens. In most subjects, CD4+ precursors of CTL were distinguished not only by their lack of Leu 8 expression but also by their relatively high density of CD2, LFA-1, and LFA-3, molecules known to mediate non-specific cell-to-cell adhesion and postulated to be markers of immunologic memory. The absence of Leu 8 does not appear to be a reliable memory cell marker, however, because Leu 8+ as well as Leu 8-, CD4+ cells from PPD skin test positive subjects responded to the recall Ag, PPD. During 3 mo of continuous culture with allogeneic stimulators, Leu 8- cells retained their cytolytic activity and remained unreactive with anti-Leu 8 mAb, whereas Leu 8+ cells remained non-cytolytic and reactive with anti-Leu 8, suggesting that under the conditions used the Leu 8 phenotype is relatively stable. PHA or anti-CD3 mAb enhanced non-specific killing by alloantigen-stimulated CD4+,Leu 8- lines but failed to unmask any cytolytic potential in CD4+,Leu 8+ lines. We conclude that MHC class II-specific cytolytic CD4+ T cells can be distinguished from non-cytolytic CD4+ cells on the basis of their surface phenotype, and that most CD4+ CTL are contained within the Leu 8- subpopulation.